Engineering microbial strains with superior production capabilities is one of the most challenging and intriguing endeavors in bioprocess development. However, due to the lack of rational and systematic approaches, the timelines and cost for strain development can be large and at times prohibitive for many products. In this STTR, we will utilize an integrated computational/experimental platform centered upon constraint-based modeling to rationally engineer microbial strains for enhanced isoprenoid production. Specifically, Phase I will focus on enhancing the production of amorphadiene, an immediate precursor to the powerful natural antimalarial drug artemisinin, in Escherichia coli. First, promising metabolic engineering targets will be identified using state-of-the-art computational approaches in constraint-based modeling. One approach will rely on the OptKnock framework to elucidate sets of gene deletions predicted to cause obligatory funneling of carbon into the isoprenoid pathway during the growth phase. Another approach will center upon the minimization of metabolic adjustment (MOMA) methodology to find deletions leading to increased amorphadiene production if the metabolic fluxes in the mutant organisms undergo a minimal redistribution from their parental counterpart. The identified deletions will be implemented in an E. coli strain engineered to produce amorphadiene as the sole isoprenoid product. In addition, adaptive evolution will be applied to strains designed by OptKnock to exhibit growth-coupled production. The designed strains will then be characterized using batch fermentations and various process variables (e.g., growth rate, oxygen and substrate uptake rates, product/byproduct production rates) will be measured. Lastly, the experimental findings will be reconciled with the original predictions to gauge the overall success of the combined modeling/experimental platform. In subsequent phases of the project, we will target several other isoprenoids of biotechnological and biomedical importance with the goal of generating at least one industrially competitive production strain. This program will lead to the development of a systematic approach to metabolic engineering that leverages genomic information and a host of experimental data for the rational design of production hosts. The developed technology will significantly expedite and lesson the cost of strain development for the production of multiple therapeutic compounds. The ultimate goal of this work is to develop an integrated computational/experimental platform for improving the microbial production of isoprenoid-based drugs and drug precursors. Microbial production of isoprenoids represents a favorable alternative to chemical extraction or synthesis as these compounds are typically found in extremely small quantities in nature and their synthesis is often expensive and inefficient. This project will first focus on improving the production of amorphadiene, an immediate precursor to the powerful natural antimalarial drug artemisinin, in Escherichia coli, and then progress towards other isoprenoids of biotechnological and biomedical importance. [unreadable] [unreadable] [unreadable]